Vaccines or drugs in solution ready for injection are inherently unstable and need refrigeration. The pharmaceutical industry has traditionally tackled the instability problem by freeze-drying drugs. This is expensive, inconvenient and inherently dangerous, since incorrect reconstitution of dried drugs can result in wrong doses or contaminated solutions. Many attempts have been made over the past 100 years to develop robust, stable, ready-to-inject liquid formulations with pitiful lack of success. Only inherently tough small molecule drugs can survive in aqueous solution with a useful shelf life.
This problem is particularly acute in the vaccine industry. By the year 2005 it is estimated that 3.6 billion doses of vaccine will have to be administered world-wide. It has been stated by the World Health Organization (WHO) that this will not be possible using standard vaccine formats which need to be refrigerated at all times ("Revolutionizing Immunizations." Jodar L., Aguado T., Lloyd J. and Lambert P-H. Genetic Engineering News Feb. 15, 1998). A "cold chain" of refrigerators is currently in use, which stretches from the vaccine factories to provincial towns in the developing world. The cost of the cold chain for the vaccine industry and for non-governmental health organizations running immunization campaigns is enormous. The WHO has estimated that just the maintenance cost of the cold chain is over $US 200 million annually. In addition, immunization campaigns may reach only those living close to the last link of the cold chain.
Vaccination campaigns require medically trained staff to ensure that the dose is correctly injected and shows no signs of degradation. The need to reconstitute some vaccines, such as measles, yellow fever and BCG, in the field is also a serious concern. This must be done precisely to ensure correct dosage and it also introduces a potential source of contamination, which has frequently led to clinical disasters. In addition, it is often necessary to give more than one vaccine at a session and this may require multiple injections, as particular mixtures or "multivalent" vaccines may not be available due to the chemical incompatibility of some of the components. The WHO has highlighted these problems by actively encouraging research into the next generation of stable vaccines which have no need for refrigeration and which need no reconstitution ("Pre-Filled Monodose Injection Devices: A safety standard for new vaccines, or a revolution in the delivery of immunizations?" Lloyd J. and Aguado M. T. WHO publication May 1998. "General policy issues: injectable solid vaccines: a role in future immunization?" Aguado M. T., Jodar L., Lloyd J., Lambert P. H. WHO publication No A59781.)
An ideal solution to this problem would be completely stable, ready-to-inject formulations. Such stable vaccines could be packed as individual doses in an injecting device itself, or, for mass immunization campaigns, shipped in larger volumes and administered by means of a needle-free jet injector. The transdermal delivery of dry solids by gas jet injection has been described (Sarphie D F, Burkoth T L. Method for providing dense particle compositions for use in transdermal particle delivery. PCT Pub No. WO 9748485 (1996)) and transdermal vaccination with dry DNA vaccines is apparently very effective ("Powderject's Hepatitis B DNA Vaccine First To Successfully Elicit Protective Immune Response In Humans" at http://www.powderject.com/pressreleases.htm (1998)).
The hypersonic shockwave of helium gas that is used to drive these powder injectors has a limited power and cannot deliver its dose of fine particles intra-muscularly. This is because the low-mass particles cannot achieve adequate momentum for deep penetration. While the intradermal delivery of DNA vaccines coated on to colloidal gold particles is adequate for good immunogenicity, the common vaccines, adjuvanted with insoluble aluminum or calcium salts, induce unacceptable skin irritation. They must be given intramuscularly. What is required is a flexible system capable of a range of delivery depths, from intradermal to deep intramuscular, similar to that achievable by existing needle and syringe technology. For mass vaccination campaigns this has been solved by the development of the liquid jet injector capable of accelerating a narrow (.about.0.15 mm diameter) stream of liquid, using pressures of around 3,000 psi, into a "liquid nail". This device delivers its dose painlessly through the skin into the deep subcutaneous or muscle tissue by punching a minute hole through the epidermis. The high momentum imparted to the liquid stream ensures deep penetration. To date, the injected drugs and vaccines have been water-based but because of the instability problems discussed above, the range of stable aqueous products accessible to this technology is very limited.
It is now recognized that a wide range of bioactive molecules may be stabilized by drying in sugar glasses (Roser B. "Protection of proteins and the like" UK patent No 2,187,191. Roser B and Colaco C. "Stabilization of biological macro-molecular substances and other organic compounds" PCT Pub No WO 91/18091. Roser B. and Sen S. "New stabilizing glasses". PCT patent Application no: 9805699.7. 1998). These dry, stabilized actives are unaffected by hostile environments such as high temperatures and ionizing radiation.
The mechanism underlying the remarkable stabilization of molecules by sugars is glass-transformation. As the sugar solution containing an active molecule is dried, it can either crystallize when the solubility limit of the sugar is reached, or can become a supersaturated syrup. The ability of the sugar to resist crystallization is a crucial property of a good stabilizer. Trehalose is good at this (Green J L. & Angel C A. Phase relations and vitrification in saccharide water solutions and the trehalose anomaly J. Phys. Chem. 93 2880-2882 (1989)) but not unique. Further drying progressively solidifies the syrup, which turns into a glass at a low residual water content. Imperceptibly, the active molecules change from liquid solution in the water to solid solution in the dry sugar glass. Chemical diffusion is negligible in a glass and therefore chemical reactions virtually cease. Since denaturation is a chemical change it cannot occur in the glass and the molecules are stabilized. In this form the molecules can remain unchanged providing one other condition is met. This is the second crucial property of a good stabilizer viz. that it is chemically inert and non-reactive. Many glasses fail because they react with the product on storage. Obvious problems occur with reducing sugars, which may form good physical glasses but then their aldehyde groups attack amino groups on the products in a typical Maillard reaction. This is the main reason that many freeze-dried pharmaceuticals require refrigerated storage. Non-reactive sugars give stable products, which require no refrigeration at all.
Biomolecules immobilized in sugar glass are also stable in non-aqueous industrial solvents in which they themselves and the sugar are both insoluble (Cleland J L. and Jones A J S. "Excipient stabilization of polypeptides treated with organic solvents" U.S. Pat. No. 5,589,167. (1994)). Since the sugar glass acts as an impermeable barrier in a non-solvent liquid, the biomolecules in solid solution in the glass are protected both from the chemical reactivity of the solvent and from the environment. Providing the liquid itself is stable, sensitive products in suspended glass particles constitute a stable two phase liquid formulation. Industrial solvents of the kind described by Cleland and Jones (1994) have a limited utility in processing. Substituting a bio-compatible non-aqueous liquid would enable stable liquid formulations of even the most unstable drugs, vaccines and diagnostics to be formulated.
The first generation of stable non-aqueous liquids designed to be used in drug or vaccine delivery (B. J. Roser and S. D. Sen "Stable particle in liquid formulations". PCT Patent Application no. GB98/00817 described formulations of powders of stabilizing glasses containing the active, suspended in injectable oils such as sesame, arachis or soya oil or simple esters such as ethyl oleate. The suspended sugar glass particles are of an intensely hydrophilic nature while the oils are hydrophobic. Because of the strong tendency of the hydrophilic and hydrophobic phases to separate, the sugar glass particles tended to clump together. In order to stabilize such "water in oil" type suspensions the use of oil-soluble surfactants dissolved in the continuous oil phase was often required.
These low HLB (Hydrophilic/Lipophilic Balance) surfactants accumulate at the interface between the hydrophilic particles and the oil and coat them with an amphiphilic layer which is more compatible with the continuous oil phase. Because each sugar glass particle is separated from its neighbors by dry oil, no chemical interaction can go on between particles. It is therefore possible to have several different populations of particles, each containing a different potentially interactive molecule, in the same oil preparation, without them being able to interact. Complex multivalent vaccines can be produced in this way.
However, this approach has been subsequently found to have certain drawbacks that prevent it from being a universal solution. These include the inevitable sedimentation of the suspended particles, which have a typical density around 1.5 g/cm.sup.3, in the less dense, oily vehicle. The patent acknowledges this problem and aims to solve it by reducing the particle size to below 1 .mu.m in diameter in order for them to remain suspended by thermodynamic forces such as Brownian motion. The requirement for all particles to be below 1 .mu.m in diameter is a disadvantage of the proposed formulations. Achieving such small particle powders is by no means an easy task. Improved spray drier designs may be able to achieve this but the small particle size would prevent the use of cyclone type collectors and require a system of filters for product recovery.
Reducing particles to sub-micron size may also, in theory, be achieved after the particles are suspended in the oil, with high-pressure micro-homogenizing equipment such as the Microfluidizer (Constant Systems Inc.). This involves an extra step to the process and we have found it not to be very efficient in breaking down spray-dried sugar glass microspheres, which have very high mechanical strength because of their spherical shape. This mandates multiple passes through the equipment. Even then, this tends to leave a number of the larger particles untouched and therefore would require a subsequent filtration or sedimentation step to remove them. Also, the high viscosity of the suspensions in the usual oily vehicles makes them difficult both to draw up into the syringe and requires that they be injected slowly. It precludes fast flows through fine nozzles such as are experienced in a liquid jet injector system.
It has also been found that particles suspended in an oil, specially when containing a low HLB surfactant, are difficult to extract subsequently into an aqueous environment because, surprisingly, they maintain a tightly bound, water repellent coat of oil around them, even after washing in aqueous buffer. They therefore require very vigorous shaking and mixing or the addition of yet more water-soluble detergent (this time with a high HLB) for the particles to leave the oil phase and enter the water phase. This becomes more of a problem as the particle size is reduced. The final outcome is often a rather messy mixed emulsion rather than two cleanly separate phases. In the body this problem can cause slow and unpredictable release of the active rather than the prompt and predictable delivery required. Extraction in vitro into an aqueous environment results in the oil floating on top of the aqueous phase containing the dissolved active. This may not be acceptable for certain in vitro applications such as diagnostic kits or automated assay systems. Finally, most of the natural, FDA-approved, oils, which can be used clinically, are vulnerable to photodegradation, oxidation or other forms of damage and require careful storage in the dark at relatively low temperatures. Additionally, they are not completely chemically inert so that they can slowly react with the suspended particles.
The Alliance Pharmaceutical Company has explored the use of powders of water-soluble substances in the remarkable new non-aqueous perfluorocarbon liquids (Kirkland WD Composition and method for delivering active agents. U.S. Pat. No. 5,770,181. (1995)). This patent is primarily concerned with the function of the PFCs as oral contrast enhancing agents for diagnostic imaging of the intestines. The water-soluble powders exemplified therein were added to improve the palatability or the enhancement of the contrast effect in the gastrointestinal tract of the PFCs. However, Kirkland perceptively realized that these liquids could also be used for drug delivery although there are no examples given. In particular, only shelf stable commercially available powders are exemplified in the patent. We have now found that fragile actives stabilized in sugar glass microspheres can be engineered to produce extremely stable two-phase PFC liquid formulations for both oral and parenteral delivery. This greatly extends the utility of the Kirkland patent to the delivery of parenteral drugs and vaccines in ready-to-inject formulations that require no refrigeration of any kind. Of particular value is the discovery that the low viscosity, high density and low surface tension of PFCs means that these stable suspensions can be delivered by automatic devices such as liquid jet injectors. This opens up two important additional fields to this technology namely mass immunization campaigns and also self injection.
Perfluorocarbons (PFCs) are novel, extremely stable liquids produced by the complete fluorination of certain organic compounds. They cannot be classified as either hydrophilic or lipophilic, as they are in fact essentially immiscible with both oil and water or any other solvent whether polar or non-polar, except other PFCs. (Reviewed in Krafft M P & Riess J G. "Highly fluorinated amphiphiles and colloidal systems, and their applications in the biomedical field. A contribution." Biochimie 80 489-514 1998). Furthermore, they do not participate in hydrophobic interactions with oils nor hydrophilic interactions with water or hydrophilic materials. As a consequence gross phase separation, as seen when hydrophilic particles clump strongly together in oil, tends not to occur in PFCs. They may not require surfactants to produce stable suspensions, but fluorohydrocarbon (FHC) surfactants are available (Krafft & Riess 1998) and are active at minute concentrations in PFC liquids. At these very low concentrations FHC surfactants can ensure perfect monodisperse systems of certain particles which show a tendency to aggregate in their absence. The PFC liquids themselves are chemically completely non-reactive and the lower molecular weight types do not accumulate in the body but, being volatile, are eventually exhaled in the breath.
Because they are excellent solvents for gases, PFCs have already been used in large quantities in very special clinical applications. Their ability to exchange carbon dioxide for dissolved oxygen is better than that of haemoglobin. This was first demonstrated in "bloodless rats" by R. P. Geyer in 1968 (Geyer R P, Monroe R G & Taylor K. "Survival of rats totally perfused with perfluorocarbon-detergent preparation." in: Organ Perfusion and Preservation, J. V Norman, J Folkman, L. E. Hardison, L. E Ridolf and F. J. Veith eds. Appleton-Century-Crofts, New York. 85-95 (1968)). Perfluorooctyl bromide, in the form of a PFC-in-water emulsion and under the trade name Oxygent.TM. (Alliance Pharmaceutical Corp.) is presently being evaluated in humans as an alternative to blood transfusion for certain surgical procedures. PFCs have also been used by inhalation, as liquids, into the lungs as a treatment for respiratory distress syndrome in premature babies.
Their high density combined with chemical inertness has also been found to be valuable. Perfluorophenanthrene, under the trade name Vitreon.TM. (Vitrophage Inc.), is used to prevent collapse of the capsule of the eye during surgery and to permit repositioning of detached retinas. PFCs have also been used as contrast media for Magnetic Resonance Imaging (MRI) and for this purpose it has been reported that hydrophilic powders may be suspended in them in order to either improve their imaging properties or make them more palatable. (Kirkland W. D. "Composition and method for delivering active agents" U.S. Pat. No. 5,770,181. 1998). This patent also suggests the use of PFCs as the continuous phase for delivering particulate water-soluble drugs. Since the number of parenteral drugs, which are stable as dry powders at room temperature is limited, this patent does not have applicability to the majority of injectable drugs. However, the combination of drug stabilization in microsphere powders of sugar glasses as described in Roser and Garcia de Castro (1998) and injectable PFCs renders this technology applicable to virtually all parenteral drugs and vaccines.